Difluoroacetic acid ion pairing reagent for high sensitivity, high resolution LC-MS of biomolecules

ABSTRACT

The present disclosure relates to the determination of analytes in a sample using chromatography. The present disclosure provides methods of separating an analyte from a sample. A mobile phase is flowed through a chromatography column. The mobile phase includes about 0.005% (v/v) to about 0.20% (v/v) difluoroacetic acid and less than about 100 ppb of any individual metal impurity. A sample including the analyte is injected into the mobile phase. The analyte is separated from the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationNo. 62/623,059 filed on Jan. 29, 2018, entitled “Difluoroacetic Acid IonPairing Reagent for High Sensitivity, High Resolution LC-MS ofProteins,” the entire contents of which is hereby incorporated herein byreference.

FIELD OF THE TECHNOLOGY

The present disclosure relates to methods of separating an analyte froma sample. More particularly, the present disclosure relates to the useof high purity difluoroacetic acid as a mobile phase in high resolutionliquid chromatography-mass spectrometry to separate and detect ananalyte from a sample.

BACKGROUND

Mass spectrometry (“MS”) is an analytical technique that measures themass-to-charge ratio of a charged molecule or molecule fragments formedfrom a sample. MS is used to analyze the mass, chemical composition,and/or chemical structure of a sample of interest. In general, MSincludes three steps: ionizing a sample to form charged molecules ormolecule fragments (i.e., ions); separating the ions according to theirmass-to-charge ratio; and detecting the separated ions to form amass-to-charge signal (i.e., spectra). The formation of the ions can beachieved through routine MS ionization techniques, such as, for example,Electrospray ionization (ESI), Fast Atom Bombardment (FAB), ChemicalIonization (CI), Electron Impact (EI), Atmospheric Solids AnalysisIonization (ASAI), Atmospheric Pressure Photoionization (APPI),Desorption Electrospray Ionization (DESI), Atmospheric Pressure VaporSource (APVS), or Matrix-Assisted Laser Desorption/Ionization (MALDI).

There are many different types of MS devices. For example, sector,time-of-flight, quadrupole, ion trap, Fourier transform ion cyclotronresonance, and tandem (two or more of the above combined in tandem ororthogonal) mass spectrometers are all different instruments that areconsidered to be MS devices. Certain characteristics of MS analysisinclude, e.g., mass accuracy, resolution, sensitivity, dynamic range,selectivity, and specificity, etc.

Protein reversed phase liquid chromatography (“RPLC”) is heavilydependent on the conditions under which it is performed. The bestresolving power has, to date, only been achieved through the use of ionpairing with a hydrophobic, acidic mobile phase additive, liketrifluoroacetic acid (“TFA”). However, TFA is known to suppresselectrospray ionization mass spectrometry signals and to complicate MSspectra with interference due to its ability to form gas-phase ion pairswith positively-charged analyte ions. Consequently, a weaker ion pairingadditive, formic acid, has been preferred by some. However, with formicacid mobile phases, protein RPLC methods fail to approach their optimalresolving power.

Several alternative mobile phase additives have been proposed over theyears. In 2002, Monroe and co-workers proposed the use ofmonofluoroacetic acid for peptide LC-MS, showing that it could affordslightly better chromatography than formic acid and better ion yieldsversus TFA. Monroe, M. E., Development of Instrumentation andApplications for Microcolumn Liquid Chromatography Coupled toTime-of-Flight Mass Spectrometry, The University of North Carolina atChapel Hill, 2002. Serious concerns were, however, raised over the useof any monohalogenated acids given their acute toxicity and ability todisrupt the citric acid cycle. In pursuit of another viable alternativeacid modifier, Monroe and co-workers considered the use of DFA, butfailed to pursue additional work. Id.

Yamamoto et al. explored the use of DFA for the reversed phaseseparation and MS detection of small molecule, basic analytes, such asimipramine and nadolol. Yamamoto, E. et al., Application of partiallyfluorinated carboxylic acids as ion pairing reagents in LC/ESI-MS,Talanta 2014, 127, 219-24. The analysis demonstrated that DFA is nearlyas effective as TFA in increasing chromatographic retention of analytesbut that it can do so without significant detriment to ionization. Id.

Wagner et al., published on the effects of a one-to-one exchange of TFAfor DFA and its resultant use for protein RPLC at a conventional mobilephase modifier concentration of 0.1% (v/v). Wagner, B. M., et al, Toolsto Improve Protein Separations, LCGC North America, 2017, 33 (11),856-865. Wagner and co-workers showed that similar chromatographicresults could be obtained for an intact monoclonal antibody using eithermethod condition. Id. Wagner suggested, but did not show, that DFA couldbe a more MS friendly methodology.

SUMMARY

What is needed is a mobile phase additive for use in biomolecule (e.g.,proteins, peptides, and/or glycans and more specifically for thecharacterization of protein therapeutics such as monoclonal antibodies(mAb) and antibody drug conjugates (ADC)) LC-MS that solves the problemsassociated with the use of TFA, namely that TFA is known to suppresselectrospray ionization mass spectrometry signals and complicate massspectra due to its ability to form gas-phase ion pairs withpositively-charged analyte ions, but that also maintains optimalresolving power of the chromatography. The mobile phaseadditive/modifier should not only achieve high sensitivity detection,but should also provide gains in the resolution of the chromatography.This technology solves the challenge with biomolecule LC-MS analysesthat exists in achieving a balanced optimization between highchromatographic resolution, high mass spectrometric sensitivity, andmass spectral quality (namely the lack of undesired ion adducts).

To solve the problems related to the use of TFA in biomolecule (e.g.,protein, peptide, and/or glycan) LC-MS (e.g., RPLC), the technologyprovides methods based on low concentrations of an alternative acid,difluoroacetic acid (“DFA”), used with or without phenyl-basedstationary phases that are capable of yielding unforeseen optimizationof both chromatographic and mass spectrometric performance. Thetechnology also includes a composition of DFA having low level metalimpurities, for example, less than about 100 ppb of any individual metalimpurity. The purity of DFA leads to the ability to achieve massspectrometry (MS) quality sufficient for a DFA based liquidchromatography-mass spectrometry (“LC-MS”) method to produceinterpretable data. The technology also includes the use of otherhalogenated acids, for example, dichloroacetic acid or dibromoaceticacid. Monohalogenated acids (e.g., monofluoroacetic acid ormonochloroacetic acid) can also be used, but are acutely toxic due totheir ability to be metabolized as part of the citric acid cycle.

The use of DFA has not yet become routine in biomolecule LC-MS. Currentsources of DFA have been found to contain high sodium and potassiumconcentrations. These salt adducts do not adversely affect separations,but they do disrupt the interpretability of mass spectra. Thus an LC-MStechnique was developed that used purified DFA with an unprecedentedbalance of chromatographic resolution and MS sensitivity. As shownherein, purified DFA affords higher MS sensitivity than TFA and itprovides better chromatographic resolution. These gains in analyticalcapabilities amount to a new LC-MS platform suitable for subunit-levelcharacterization of mAb-based therapeutics, including even a highlyhydrophobic cysteine-linked ADC. Unlike previous methods, this purifiedDFA based RPLC-MS method shows little to no on-column sampledegradation, complete analyte recovery, noteworthy proteoformresolution, and 3-fold higher MS sensitivity, which in sum makes itpossible to detect and monitor trace-levels of product-relatedimpurities with higher fidelity.

The present disclosure relates to a method of separating an analyte froma sample. The method includes flowing a mobile phase through achromatography column. The mobile phase includes about 0.005% (v/v) toabout 0.20% (v/v) difluoroacetic acid and less than about 100 ppb of anyindividual metal impurity. A sample including the analyte is injectedinto the mobile phase. The analyte is separated from the sample. Themethod can include one or more of the embodiments described herein.

The present disclosure also relates to a method of separating an analytefrom a sample. The method includes flowing a mobile phase through achromatography column. The mobile phase includes about 0.005% (v/v) toabout 0.20% (v/v) difluoroacetic acid. A sample including the analyte isinjected into the mobile phase. The analyte is separated from thesample. The analyte is detected with a mass spectrometer. The massspectrometer produces a mass spectrum having less than about 5% relativeion intensity corresponding to metal or salt adducts. The method caninclude one or more of the embodiments described herein.

The chromatography column can be a liquid chromatography column or areversed phase chromatography column. The chromatography column can be ahydrophilic interaction chromatography (“HILIC”) column. Thechromatography column can include a stationary phase having aphenyl-based surface chemistry. The stationary phase can be asuperficially porous silica stationary phase bonded with phenylmoieties. The stationary phase can be a fully porous silica stationaryphase bonded with phenyl moieties. In some embodiments, the stationaryphase is an organosilica stationary phase bonded with phenyl moieties.The chromatography column can include a stationary phase having apolymeric polystyrene divinyl benzene surface chemistry.

The mobile phase can have less than about 50 ppb of any individual metalimpurity. The mobile phase can have less than about 20 ppb of anyindividual metal impurity. The mobile phase can have less than about 90ppb, 80 ppb, 70 ppb, 60 ppb, 50 ppb, 40 ppb, 30 ppb, 20 ppb, or 10 ppbof any individual metal impurity. The individual metal impurity, can be,for example, sodium, potassium, calcium, iron, or combinations thereof.

The mobile phase can have about 0.01% (v/v) to about 0.05% (v/v)difluoroacetic acid. The mobile phase can have about 0.02% (v/v) toabout 0.05% (v/v) difluoroacetic acid. The mobile phase can have about0.01% (v/v) to about 0.02% (v/v) difluoroacetic acid. The mobile phasecan also include water, acetonitrile, methanol, propanol, butanol,pentanol, or combinations thereof.

The analyte can be a biomolecule. The analyte can be a protein, apeptide, a glycan or a combination thereof. The analyte can includemultiple proteins, multiple peptides, multiple glycans or combinationsthereof.

The method can also include determining the molecular weight of theanalyte, e.g. a biomolecule. For example, the method can includedetermining the molecular weight of the protein, the peptide, theglycan, or combinations thereof.

The method can also include detecting the analyte with a massspectrometer. Analyte ions can be generated by the mass spectrometer.The analyte ions can be generated by electrospray ionization ordesorption electrospray ionization. A mass spectrum of the analyte ionscan be acquired.

The present disclosure also relates to a kit. The kit includes achromatography column having a stationary phase material containedinside the column. The kit also includes an ampoule having a volume ofmobile phase additive. The mobile phase additive includes difluoroaceticacid and less than about 100 ppb of any individual metal impurity. Thekit also includes instructions. The instructions instruct the user toobtain a sample containing at least one biomolecule (e.g., a protein, apeptide, a glycan or any combination) in a sample matrix, dilute themobile phase additive with a solvent to obtain about 0.005% (v/v) toabout 0.20% (v/v) difluoroacetic acid, flow the sample with the dilutedmobile phase through the column to substantially resolve and retain theat least one biomolecule (e.g., a protein, a peptide, a glycan or anycombination), and detect the at least one biomolecule using a detector.The kit can include one or more of the embodiments described herein.

The stationary phase material can be a superficially porous silicastationary phase bonded with phenyl moieties. The stationary phase canbe a fully porous silica stationary phase bonded with phenyl moieties.In some embodiments, the stationary phase is an organosilica stationaryphase bonded with phenyl moieties. The stationary phase material can bea polymeric polystyrene divinyl benzene surface chemistry.

The present disclosure relates to a kit that includes a chromatographycolumn having a stationary phase material contained inside the columnand a container having a volume of mobile phase. The mobile phase hasabout 0.005% (v/v) to about 0.20% (v/v) difluoroacetic acid and lessthan about 100 ppb of any individual metal impurity. The kit alsoincludes instructions for obtaining a sample containing at least onebiomolecule (e.g., a protein, a peptide, a glycan or any combination) ina sample matrix, flowing the sample with the mobile phase through thecolumn to substantially resolve and retain the at least one biomolecule(e.g., a protein, a peptide, a glycan or any combination), and detectingthe at least one biomolecule using a detector. The kit can include oneor more of the embodiments described herein.

The stationary phase material can be a superficially porous silicastationary phase bonded with phenyl moieties. The stationary phase canbe a fully porous silica stationary phase bonded with phenyl moieties.In some embodiments, the stationary phase is an organosilica stationaryphase bonded with phenyl moieties. The stationary phase material can bea polymeric polystyrene divinyl benzene surface chemistry.

The present disclosure also relates to a method of purifyingdifluoroacetic acid containing greater than 100 ppb of an impurity. Theimpurity can be sodium, potassium, calcium, iron, or combinationsthereof. The method includes distilling the difluoroacetic acid toobtain a high-purity difluoroacetic acid containing less than 100 ppb ofthe impurity. The method can include one or more of the embodimentsdescribed herein.

The high-purity difluoroacetic acid can contain less than 50 ppb of theimpurity. The high-purity difluoroacetic acid can contain less than 20ppb of the impurity. In some embodiments, the high-purity difluoroaceticacid can contain less than 40 ppb, less than 30 ppb, or less than 10 ppbof the impurity.

The present disclosure provides a number of advantages over currentsystems and methodology. For example, purified DFA provides a moreMS-friendly alternative to TFA for biomolecule (e.g., a protein, apeptide, a glycan or any combination) LC-MS. In addition, purified DFAprovides gains in the resolution of the chromatography as compared toTFA.

BRIEF DESCRIPTION OF THE DRAWINGS

The technology will be more fully understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1A is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm BioResolve RP mAb Polyphenyl450 Å 2.7 μm column with 0.1% TFA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 1B is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm BioResolve RP mAb Polyphenyl450 Å 2.2 μm column with 0.1% FA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 1C is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm AMT Halo Protein C4 400 Å3.4 μm column with 0.1% TFA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 1D is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm AMT Halo Protein C4 400 Å3.4 μm column with 0.1% FA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 1E is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm Agilent AdvanceBio RP-mAbDiphenyl 450 Å 3.5 μm column with 0.1% TFA mobile phase modifier,according to an illustrative embodiment of the technology.

FIG. 1F is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm Agilent AdvanceBio RP-mAbDiphenyl 450 Å 3.5 μm column with 0.1% FA mobile phase modifier,according to an illustrative embodiment of the technology.

FIG. 1G is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm Acquity UPLC Protein BEH C4300 Å 1.7 μm column with 0.1% TFA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 1H is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm Acquity UPLC Protein BEH C4300 Å 1.7 μm column with 0.1% FA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 1I is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm BioResolve RP mAb Polyphenyl450 Å 2.7 μm column with 0.01% DFA mobile phase modifier, according toan illustrative embodiment of the technology.

FIG. 1J is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm BioResolve RP mAb Polyphenyl450 Å 2.7 μm column with 0.02% DFA mobile phase modifier, according toan illustrative embodiment of the technology.

FIG. 1K is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm BioResolve RP mAb Polyphenyl450 Å 2.7 μm column with 0.1% DFA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 1L is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm AMT Halo Protein C4 400 Å3.4 μm column with 0.01% DFA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 1M is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm AMT Halo Protein C4 400 Å3.4 μm column with 0.02% DFA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 1N is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm AMT Halo Protein C4 400 Å3.4 μm column with 0.1% DFA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 1O is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm Agilent AdvanceBio RP-mAbDiphenyl 450 Å 3.5 μm column with 0.01% DFA mobile phase modifier,according to an illustrative embodiment of the technology.

FIG. 1P is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm Agilent AdvanceBio RP-mAbDiphenyl 450 Å 3.5 μm column with 0.02% DFA mobile phase modifier,according to an illustrative embodiment of the technology.

FIG. 1Q is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm Agilent AdvanceBio RP-mAbDiphenyl 450 Å 3.5 μm column with 0.1% DFA mobile phase modifier,according to an illustrative embodiment of the technology.

FIG. 1R is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm Acquity UPLC Protein BEH C4300 Å 1.7 μm column with 0.01% DFA mobile phase modifier, according toan illustrative embodiment of the technology.

FIG. 1S is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm Acquity UPLC Protein BEH C4300 Å 1.7 μm column with 0.02% DFA mobile phase modifier, according toan illustrative embodiment of the technology.

FIG. 1T is a chromatogram for reduced, IdeS digested NIST referencematerial 8671 as observed using a 2.1×50 mm Acquity UPLC Protein BEH C4300 Å 1.7 μm column with 0.1% DFA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 2A is a graph showing effective peak capacity values for reduced,IdeS digested NIST reference material 8671 as observed using various2.1×50 mm columns and 0.1% TFA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 2B is a graph showing effective peak capacity values for reduced,IdeS digested NIST reference material 8671 as observed using various2.1×50 mm columns and 0.01% DFA mobile phase modifier. according to anillustrative embodiment of the technology.

FIG. 2C is a graph showing effective peak capacity values for reduced,IdeS digested NIST reference material 8671 as observed using various2.1×50 mm columns and 0.02% DFA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 2D is a graph showing effective peak capacity values for reduced,IdeS digested NIST reference material 8671 as observed using various2.1×50 mm columns and 0.1% DFA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 2E is a graph showing effective peak capacity values for reduced,IdeS digested NIST reference material 8671 as observed using various2.1×50 mm columns and 0.1% FA mobile phase modifier, according to anillustrative embodiment of the technology.

FIG. 3A is a graph showing LC-MS sensitivity for the detection of thelight chain subunit of reduced, IdeS digested NIST reference material8671 as observed using an LC column commercially available from WatersCorporation (Milford, Mass.) under the tradename BioResolve™ RP mAbPolyphenyl 2.1×50 mm column and a detector commercially available fromWaters Corporation (Milford, Mass.) under the tradename ACQUITY® QDa®single quadrupole mass detector. FIG. 3A shows the total ionchromatogram (TIC) peak heights resulting from the use of various mobilephase modifiers, according to an illustrative embodiment of thetechnology.

FIG. 3B is a graph showing LC-MS sensitivity for the detection of thelight chain subunit of reduced, IdeS digested NIST reference material8671 as observed using an LC column commercially available from WatersCorporation (Milford, Mass.) under the tradename BioResolve™ RP mAbPolyphenyl 2.1×50 mm column and a detector commercially available fromWaters Corporation (Milford, Mass.) under the tradename ACQUITY® QDa®single quadrupole mass detector. FIG. 3B shows TIC signal-to-noiseresulting from the use of various mobile phase modifiers, according toan illustrative embodiment of the technology.

FIG. 4A is a deconvoluted ESI mass spectra for the light chain subunitof NIST reference material 8671 as observed using 0.1% (v/v) TFA with amass spectrometer commercially available from Waters Corporation(Milford, Mass.) under the tradename Synapt® G2-Si. The relativeintensities of undesirable ion adducts, including Na and K, arereported, according to an illustrative embodiment of the technology.

FIG. 4B is a deconvoluted ESI mass spectra for the light chain subunitof NIST reference material 8671 as observed using 0.1% (v/v) DFA with amass spectrometer commercially available from Waters Corporation(Milford, Mass.) under the tradename Synapt® G2-Si. The relativeintensities of undesirable ion adducts, including Na and K, arereported, according to an illustrative embodiment of the technology.

FIG. 5 is a chart showing metal impurities quantified by inductivelycoupled plasma (ICP)-MS in a sample of DFA (Oakwood, part number 001231,lot D06N). Concentrations are reported in part per billion (“ppb”),according to an illustrative embodiment of the technology.

FIG. 6A shows the influence of sodium and potassium content on passspectra quality through ICP-MS quantitation of as-received versusdistilled DFA, according to an illustrative embodiment of thetechnology. Separations performed with a BioResolve RP mAb Polyphenyl450 Å, 2.7 μm, 2.1×50 mm column using a flow rate of 0.2 mL/min, columntemperature of 80° C., and 0.25 μg mass loads.

FIG. 6B is a deconvoluted mass spectrum of the NIST mAb LC subunitobtained using as-received DFA, according to an illustrative embodimentof the technology. Separations performed with a BioResolve RP mAbPolyphenyl 450 Å, 2.7 μm, 2.1×50 mm column using a flow rate of 0.2mL/min, column temperature of 80° C., and 0.25 μg mass loads.

FIG. 6C is a deconvoluted mass spectrum of the NIST mAb LC subunitobtained using distilled DFA, according to an illustrative embodiment ofthe technology. Separations performed with a BioResolve RP mAbPolyphenyl 450 Å, 2.7 μm, 2.1×50 mm column using a flow rate of 0.2mL/min, column temperature of 80° C., and 0.25 μg mass loads.

FIG. 7A shows TIC (counts) from a cysteine-linked auristatin conjugatedantibody as separated with a C₄-bonded organosilica 300 Å fully porousstationary phase, 0.6 mL/min flow rate, 80° C. temperature, 0.1% TFAmodified mobile phases and 90:10 CAN/IPA eluent.

FIG. 7B shows total absorbtion (AU) from a cysteine-linked auristatinconjugated antibody as separated with a C₄-bonded organosilica 300 Åfully porous stationary phase, 0.6 mL/min flow rate, 80° C. temperature,0.1% TFA modified mobile phases and 90:10 CAN/IPA eluent.

FIG. 7C shows TIC (counts) from a cysteine-linked auristatin conjugatedantibody as separated with a new platform method consisting of a phenylbonded 2.7 μm superficially porous 450 Å stationary phase, 0.6 mL/minflow rate, 70° C. temperature, and 0.15% DFA modified mobile phases,according to an illustrative embodiment of the technology.

FIG. 7D shows total absorbtion (AU) from a cysteine-linked auristatinconjugated antibody as separated with a new platform method consistingof a phenyl bonded 2.7 μm superficially porous 450 Å stationary phase,0.6 mL/min flow rate, 70° C. temperature, and 0.15% DFA modified mobilephases, according to an illustrative embodiment of the technology.

FIG. 8A is a deconvoluted MS spectra of the unmodified LC fragment fromthe cysteine-linked auristatin ADC obtained using 0.1% MS-grade FAmodified mobile phase, according to an illustrative embodiment of thetechnology. Separations performed with a BioResolve RP mAb Polyphenyl450 Å, 2.7 μm, 2.1×150 mm column using a flow rate of 0.6 mL/min, columntemperature of 80° C., and 1 μg mass loads.

FIG. 8B is a deconvoluted MS spectra of the unmodified LC fragment fromthe cysteine-linked auristatin ADC obtained using 0.1% distilled DFAmodified mobile phase, according to an illustrative embodiment of thetechnology. Separations performed with a BioResolve RP mAb Polyphenyl450 Å, 2.7 μm, 2.1×150 mm column using a flow rate of 0.6 mL/min, columntemperature of 80° C., and 1 μg mass loads.

FIG. 8C is a deconvoluted MS spectra of the unmodified LC fragment fromthe cysteine-linked auristatin ADC obtained using 0.1% MS-grade TFAmodified mobile phase, according to an illustrative embodiment of thetechnology. Separations performed with a BioResolve RP mAb Polyphenyl450 Å, 2.7 μm, 2.1×150 mm column using a flow rate of 0.6 mL/min, columntemperature of 80° C., and 1 μg mass loads.

DETAILED DESCRIPTION

The present disclosure relates to methods based on low concentrations ofan alternative acid, difluoroacetic acid (“DFA”), used with or withoutphenyl-based stationary phases that are capable of yielding unforeseenoptimization of both chromatographic and mass spectrometric performance.The technology also includes a composition of DFA having low level metalimpurities, for example, less than about 100 ppb of any individual metalimpurity. The purity of DFA leads to the ability to achieve massspectrometry (MS) quality sufficient for a DFA based liquidchromatography-mass spectrometry (“LC-MS”) method to produceinterpretable data.

While the present disclosure discusses the technology in relation todifluoroacetic acid, dichloroacetic acid or dibromoacetic acid can alsobe used and are expected to show similar results with respect tounforeseen optimization of both chromatographic and mass spectrometricperformance. In addition, while the present disclosure discusses thetechnology mainly in relation to proteins, the methods can also beapplied to other biomolecules, including, for example, peptides andglycans. Moreover, the reagent and methods of the present technology arealso applicable to the analysis of peptides and the peptide mapping ofprotein therapeutics.

As used herein, the term “resolution” refers to the measure of how welltwo peaks are separated. Resolution can be determined byR_(s)=(t_(r,2)−t_(r,1))/(0.5× (w₁+w₂)), wherein t_(r) is the retentiontime of either peak 1 or peak 2, and w₁ is the peak width at half heightfor peak 1 or peak 2. In a similar fashion, resolving power and peakcapacity are used to refer to the measure of how many peaks can fitwithin a given separation space. Peak capacity can be determined asP_(c)=1+(t/w_(avg)), wherein t is the time corresponding to the givenseparation space and w_(avg) is the average peak width at half heightobserved for peaks in a given separation.

The term “protein” as used herein, refers to a polymeric chain of aminoacids called polypeptides. A protein may also include a number ofmodifications, including phosphorylation, lipidation, prenylation,sulfation, hydroxylation, acetylation, addition of carbohydrate(glycosylation and glycation), addition of prosthetic groups orcofactors, formation of disulfide bonds, proteolysis, assembly intomacromolecular complexes and the like.

As used herein, the term “about” means that the numerical value isapproximate and small variations would not significantly affect thepractice of the disclosed embodiments. Where a numerical limitation isused, unless indicated otherwise by the context, “about” means thenumerical value can vary by ±10% and remain within the scope of thedisclosed embodiments.

In concept, DFA is a more MS-friendly alternative to TFA for proteinLC-MS. DFA is 10 times less acidic than TFA, so it can be lessdetrimental to MS detection. Being a weaker acid, it is likely to formweaker ion pair interactions with analyte cations. In turn, DFA may showa lower propensity than TFA to produce adducts in mass spectra.Moreover, being composed of only two fluorine atoms, versus the three ofTFA, DFA is less likely to adsorb to materials and to be problematicallyretained throughout LC flow paths and MS ion paths, which has been anannoyance for some LC-MS scientists in their attempts to use TFA.

However, the appeal of DFA cannot be fully appreciated by speculationalone. As shown in the scope of this technology, there is more to thequality of protein LC-MS than just ionization efficiency. With thistechnology, a composition of DFA is specified that ensures theproduction of high quality protein mass spectra. This composition of DFAcan be provided in kits, along with a chromatography column andinstructions for use. In addition, novel methods are defined for proteinLC-MS that call for DFA to be used at unconventionally lowconcentrations, with or without a phenyl based stationary phase.Further, this technology provides a method of purifying commerciallyavailable difluoroacetic acid to obtain a high-purity difluoroaceticacid containing less than 100 ppb of a metal impurity.

Biomolecule (e.g., protein, peptide, and/or glycan) LC-MS is not merelyabout achieving high sensitivity detection. The capability of the methodis greatly impacted by the resolution of the chromatography.Interestingly, DFA mobile phases can actually provide gains inresolution as compared to TFA. This was a surprising result given thatformic acid, a weaker, less hydrophobic acid, leads to astoundinglylower peak capacity versus TFA. Accordingly, it was assumed that a moreacidic, more hydrophobic mobile phase additive would always yield betterchromatographic resolving power. While not limited to theory, it isbelieved that DFA is more effective than TFA in producing highresolution chromatography because it exhibits less steric bulk.Consequently, it is likely for DFA to more effectively and moreextensively interact with protein analytes and the bonded phase of anRPLC stationary phase. It might also be that the hydrophobicity of DFA,being attenuated from that of TFA, facilitates more optimal proteinadsorption, partitioning, and desorption.

FIGS. 1A-1T show the effective peak capacity values for reduced IdeSdigested NIST reference material 8671 as observed using various 2.1×50mm columns, including (1) Waters BioResolve™ RP mAb Polyphenyl, 450 Å2.7 μm; (2) AMT HALO® Protein C4, 400 Å 3.4 μm; (3) Agilent AdvanceBio®RP-mAb Diphenyl 450 Å 3.5 μm; and (4) Waters ACQUITY® UPLC®, Protein BEHC4, 300 Å 1.7 μm. FIGS. 2A-E show effective peak capacity values forreduced, IdeS digested NIST reference material 8671 as observed usingeach of the various columns of FIGS. 1A-1T. The specifics of theanalysis can be found in Example 1, herein.

As shown in an evaluation of these various column technologies, 0.1%(v/v) DFA can yield significantly higher peak capacities versus 0.1%(v/v) TFA. In particular, for separations of reduced, IdeS digested NISTreference material 8671, it has been seen that there can be increasescorresponding to up to about 40% gains in effective peak capacity. Forexample, the effective peak capacity using IdeS digested NIST referencematerial 8671 with Agilent AdvanceBio® RP-mAb Diphenyl 450 Å 3.5 μm is72.0 when using 0.1% (v/v) TFA as a mobile phase, but increases to 102.9when using 0.1% (v/v) DFA, resulting in more than a 40% gain ineffective peak capacity. In addition, the Agilent column showed about a20% gain in effective peak capacity when using 0.02% (v/v) DFA ascompared with a higher concentration, 0.1% (v/v) TFA. Moreover, therewas only a slight decrease, less than 5%, in effective peak capacitywhen 0.01% (v/v) DFA was used with the Agilent column as compared to0.1% (v/v) TFA.

As shown in FIGS. 1A-1T, and 2A-E, all chromatography columns showed anincrease in effective peak capacity when using 0.1% DFA compared to 0.1%TFA. The other three columns each showed about a 10% gain in effectivepeak capacity when using 0.1% (v/v) DFA compared to 0.1% (v/v) TFA.

These gains can also be seen when profiling samples produced by otherenzymes including IdeZ, Lys-C, and papain as well as enzymescommercially available from Genovis AB (Lund, Sweden) under thetradename FabULOUS®, FabRICATOR®, FabALACTICA® and GingisKHAN®. Evenmore notably, a superficially porous silica stationary phase bonded withphenyl moieties in a multistep silanization process has been found toprovide exemplary performance capabilities with a DFA mobile phase. Withthis stationary phase, performance is surprisingly good even with lowconcentrations of DFA additive. FIGS. 1A-1T, and 2A-E show that, indeed,when 0.01 and 0.02% DFA mobile phases are used along with this sort ofstationary phase material, exemplary resolving power is produced.However, it is not just the gain in effective peak capacity that resultswhen a superficially porous stationary phase bonded with phenyl moietiesin a multistep salinization process that provides exemplary performance,but also the percentage of phenyl coverage on the surface of thestationary phase. For example, the column commercially available fromWaters Corporation (Milford, Mass.) under the tradename BioResolve™ RPmAb Polyphenyl, has about 10% phenyl coverage making it less dependenton ion pairing. Stationary phases that can be advantageously used withthese unique conditions have been described in United States publicationno. 2018/0264438 entitled “Chromatographic Compositions” assigned toWaters Technologies Corporation, which is incorporated herein byreference in its entirety.

It is noteworthy to have discovered that an exemplary level ofchromatographic performance is possible even with the use of just 0.01%and 0.02% (v/v) DFA, because it has proven to be of benefit to thesensitivity of MS detection. FIGS. 3A and 3B show LC-MS sensitivity forthe detection of the light chain subunit of reduced, IdeS digested NISTreference material 8671 as observed using an LC column commerciallyavailable from Waters Corporation (Milford, Mass.) under the tradenameBioResolve™ RP mAb Polyphenyl 2.1×50 mm column and an MS detectorcommercially available from Waters Corporation (Milford, Mass.) underthe tradename ACQUITY® QDa® single quadrupole mass detector. FIG. 3Ashows total ion chromatogram (“TIC”) peak heights resulting from the useof various mobile phase modifiers. FIG. 3B shows TIC signal-to-noiseresulting from the use of various mobile phase modifiers. The specificsof the analysis can be found in Example 2, herein.

TIC peak height and TIC peak signal-to-noise ratio are two values thatare frequently used to define the sensitivity of an LC-MS analysis. Whenvarious mobile phase systems were employed to detect reduced, IdeSdigested NIST reference material 8671 with an MS detector commerciallyavailable from Waters Corporation (Milford, Mass.) under the tradenameACQUITY® QDa® single quadrupole mass detector, substantially differentvalues for MS sensitivity were observed. When 0.1% (v/v) DFA was used inplace of 0.1% (v/v) TFA, an approximately 4 fold increase in MSsensitivity was achieved. Furthermore, when 0.02% (v/v) DFA was used inplace of 0.1% (v/v) TFA, an approximately 8 fold increase in MSsensitivity was achieved. That a protein RPLC method is able to producenear optimal resolution under such conditions means that a new standardfor high resolution, high sensitivity LC-MS of proteins has beenestablished.

Accordingly, a method is provided for separating an analyte from asample. The analyte can be a biomolecule, for example, a protein,peptide, glycan, or combination thereof. The method includes flowing amobile phase through a chromatography column. The mobile phase can be ahalogenated acid, for example, DFA, dichloroacetic acid or dibromoaceticacid. The mobile phase can include about 0.005% (v/v) to about 0.20%(v/v) halogenated acid. In addition, the mobile phase can have less thanabout 100 ppb of any individual metal impurity. In other words, allmetal impurities in the mobile phase halogenated acid are each less than100 ppb, but combined, can be greater than 100 ppb. A sample thatcomprises the analyte is injected into the mobile phase and then theanalyte is separated from the sample.

The analyte can be separated from the sample through chromatography. Aperson having ordinary skill in the art would understand that manydifferent types of chromatography can be used with the method. Forexample, the method can be applied to liquid chromatography, RPLC,UHPLC, HPLC, and hydrophilic interaction chromatography (“HILIC”).Therefore, a liquid chromatography column, reverse phase chromatographycolumn, ultra-performance chromatography column, high-performancechromatography, and hydrophilic interaction chromatography columns canbe used in the method.

In an embodiment of this technology, a high sensitivity, high resolutionprotein RPLC method is achieved using a DFA modified mobile phase incombination with column stationary phase having a phenyl-based surfacechemistry. The chromatography column can include a stationary phasehaving phenyl-based surface chemistry. The stationary phase can beeither a fully porous or a superficially porous silica stationary phasebonded with phenyl moieties. The stationary phases can include, but arenot limited to, those found in reverse phase columns commerciallyavailable from Agilent Technologies (Santa Clara, Calif.) under thetradenames AdvanceBio® RP mAb Diphenyland Zorbax® RRHD Diphenyl, andWaters Corporation (Milford, Mass.) under the tradename BioResolve™ RPmAb Polyphenyl columns as well as the materials described in UnitedStates publication no. 2018/0264438 entitled “ChromatographicCompositions” assigned to Waters Technologies Corporation, which isincorporated herein by reference in its entirety.

In some embodiments, the stationary phase can have a polymericpolystyrene divinyl benzene surface chemistry. In another embodiment,the stationary phase can be based on organosilica bonded with phenylmoieties. These stationary phases can be found, for example, in columnscommercially available Agilent Technologies (Santa Clara, Calif.) underthe tradename PLRP-S® and from Waters Corporation (Milford, Mass.) underthe tradename ACQUITY® UPLC® BEH Phenyl, respectively.

Chromatography columns of the present technology can be used along withconcentrations of about 0.005 to about 0.20% (v/v) DFA mobile phase. Anyconcentration of mobile phase within this range can be used with thedisclosed methods, kits, and compositions. For example, a 0.01 to 0.05%(v/v) DFA mobile phase can be used with a chromatography column, e.g.,an RPLC column. In another example, a 0.02% (v/v) to 0.05% (v/v) DFAmobile phase can be used with a chromatography column, e.g., an RPLCcolumn.

In addition to chromatographic resolution, protein LC-MS methods arealso judged by the quality of the mass spectra they provide. No work hasyet been performed regarding the quality of mass spectra produced by DFAmobile phases, as evidenced by the fact that commercially availablesources of DFA produce low quality mass spectra. FIG. 4 showsdeconvoluted ESI mass spectra for the light chain subunit of NISTreference material 8671 as observed using (a) 0.1% (v/v) TFA and (b)0.1% (v/v) DFA with a mass spectrometer commercially available fromWaters Corporation (Milford, Mass.) under the tradename Synapt® G2-Si.The relative intensities of undesirable ion adducts, including Na and Kare reported. The specifics of the analysis can be found in Example 3,herein.

As shown in FIGS. 4A and 4B, a mass spectrum obtained for a light chainmonoclonal antibody (mAb) subunit using 0.1% (v/v) DFA (Oakwood, partnumber 001231, lot D06N) produced very high ion intensities for sodiated(+Na) and potassiated (+K) ions. This was a level of ion intensity(approximately 6-7%) that impaired the interpretation of the massspectrum. In contrast, a mass spectrum collected from LC-MS with a 0.1%(v/v) TFA mobile phase showed a mass spectrum of significantly greaterquality, being that the sodiated and potassiated ions accounted forrelative intensities of less than or equal to 2%. While not limited totheory, it might be possible that a particular acid have not only aneffect on formation of the analyte ions but also a differential effecton the formation of adducts. Regardless, it is reasoned that thecommercially available DFA reagents are not fit for LC-MS, because ithas not yet been realized that in order to achieve desirable massspectral quality substantially higher purity DFA must be manufactured,most specifically low metal content DFA. That is, there is a correlationbetween certain impurities and the desirable features of a protein massspectrum.

FIG. 5 shows metal impurities quantified by inductively coupled plasma(ICP)-MS in a sample of DFA (Oakwood, part number 001231, lot D06N).Concentrations are reported in part per billion (“ppb”). A sample of DFA(Oakwood, part number 001231, lot D06N) was subjected to ICP-MS toquantify its metal impurities (FIG. 5), whereby it was found that theDFA did in fact contain relatively high levels of metals, includingsodium at a concentration of 1500 ppb. Without question, this level ofmetal content is too high for it to be possible to obtain a high qualityprotein mass spectrum.

DFA-derived, deconvoluted spectra showed significant interference frompotassium and sodium adducts, as demonstrated in FIG. 6A. Incorroboration of this result, ICP-MS quantitation of the employed DFAshowed it to contain 400 ppm sodium and 2 ppm potassium (FIG. 6B).Interestingly, two other commercial sources of DFA were also confirmedto have these same problematically high concentrations (data not shown).To address this shortcoming, commercially-sourced DFA was distilled tohigher purity using an apparatus constructed from chemically-resistantperfluoroalkoxy alkane (PFA) polymer. ICP-MS results indicated that, bymeans of this distillation, sodium and potassium content of the DFAcould be reduced to a concentration of less than 20 ppb. When used forLC-MS, this distilled DFA afforded spectra with adduct levels lowered torelative intensities of only 2% (FIG. 6C).

The methods of the present technology include the use of a mobile phaseadditive that includes less than about 100 ppb of any individual metalimpurity. In other words, each metal impurity contained in the mobilephase additive is not present in an amount greater than about 100 ppb.In some embodiments, the mobile phase comprises less than about 90 ppb,80 ppb, 70, ppb, 60, ppb, 50 ppb, 40 ppb, 30 ppb, 20 ppb, or 10 ppb ofany individual metal impurity. In some embodiments, the mobile phaseadditive comprises less than about 95 ppb, 85 ppb, 75, ppb, 65, ppb, 55ppb, 45 ppb, 35 ppb, 25 ppb, or 15 ppb of any individual metal impurity.The metal impurity is any metal that affects the desirable features of amass spectrum, e.g., the quality of the mass spectrum. The metalimpurity can be, for example, sodium, potassium, calcium and/or iron.These aspects of the technology extend to any dilutions and to anymobile phases that are subsequently prepared with the above describedmobile phase additive.

The methods can include detecting the analyte with a mass spectrometer.This can be accomplished by generating analyte ions. The analyte ionscan be generated by electrospray ionization or desorption electrosprayionization.

The mass spectrometer can produce a mass spectrum having less than about5% relative ion intensity corresponding to metal or salt adducts. Themass spectrometer can produce a mass spectrum having less than about 5%relative ion intensity corresponding to metal or salt adducts when themobile phase additive includes less than about 100 ppb of any individualmetal impurity. In some embodiments, the mass spectrometer can produce amass spectrum having less than about 2%, or less than about 1%, relativeion intensity corresponding to metal or salt adducts.

The methods described herein can also include determining the molecularweight of the analyte, for example, determining the molecular weight ofthe protein, peptide, glycan, or combination thereof. Likewise, themethods described herein can be used to facilitate determining thelocation of certain post-translational modifications, as can beperformed by RPLC-MS/MS experiments.

The technology also includes methods of purifying commercially availablehalogenated acids. For example, a commercially available halogenatedacid (e.g., DFA, dichloroacetic acid or dibromoacetic acid) can beobtained. The halogenated acid contains greater than 100 ppb of a metalimpurity. The metal impurity is sodium, potassium, calcium, iron orcombinations thereof. The method includes distilling the halogenatedacid to obtain a high-purity difluoroacetic acid containing less than100 ppb of the impurity. In some embodiments, the halogenated acidcontains less than 50 ppb or less than 20 ppb of the impurity.

The commercially available halogenated acid can be purified by othermethods, for example, by filtering, centrifuging, evaporation,extraction, ion exchange, or any combination.

The technology comprises a composition of a volume of difluoroaceticacid, ranging from 10 μL to 10 mL, containing less than 100 ppb ofindividual metal impurities, including but not limited to sodium,potassium, calcium and/or iron, that is purposed for LC-MS analyses ofproteins, wherein the use of such a reagent facilitates the productionof protein mass spectra having less than 5% relative ion intensitycorresponding to metal and/or salt adducts, including but not limited tosodium, potassium, calcium and/or iron. The use of the high puritycomposition of this technology can facilitate the production of proteinmass spectra having less than 2% (and possibly even less than 1%)relative ion intensity corresponding to metal and/or salt adducts,including but not limited to sodium, potassium, calcium and/or iron. Insome embodiments, the use of the high purity composition of thistechnology can facilitate the production of protein mass spectra havingless than 1% relative ion intensity corresponding to metal and/or saltadducts, including but not limited to sodium, potassium, calcium and/oriron

The compositions of the halogenated acid can be made commerciallyavailable in the form of ready-to-use ampoules as well as a component ofa kit, such as a combined product comprised of an ampoule containing thecomposition of this technology along with an LC column or device. Forexample the kit can include a chromatography column, an ampoule having avolume of mobile phase additive, and instructions for use. Thechromatography column has a stationary phase material inside the column.The stationary phase material can be any stationary phase materialdescribed herein, for example, a superficially porous silica stationaryphase bonded with phenyl moieties, a fully porous silica stationaryphase bonded with phenyl moieties, an organosilica stationary phasebonded with phenyl moieties, or a polymeric polystyrene divinyl benzenesurface chemistry. The mobile phase additive is a halogenated acid, forexample, DFA. The mobile phase additive has less than about 100 ppb ofany individual metal impurity. The instructions instruct the user toobtain a sample containing at least one biomolecule (e.g., a protein) ina sample matrix as well as to dilute the mobile phase additive with asolvent to obtain about 0.005% (v/v) to about 0.20% (v/v) halogenatedacid (e.g., DFA). Like the DFA mobile phase additive, the solvents areto have less than about 100 ppb of any individual metal impurity, orless than about 95 ppb, 85 ppb, 75, ppb, 65, ppb, 55 ppb, 45 ppb, 35ppb, 25 ppb, or 15 ppb of any individual metal impurity. The user isthen instructed to flow the sample with the diluted mobile phase throughthe column to substantially resolve and retain the at least onebiomolecule (e.g., protein). In addition, the instructions instruct theuse to detect the at least one biomolecule (e.g., protein) using adetector.

Similarly, the composition of the halogenated acid extends toready-to-use mobile phases. A container of the ready-to-use mobile phasecan be included as part of a kit. The kit also includes a chromatographycolumn and instructions for use. The chromatography column has astationary phase material inside the column. The stationary phasematerial can be any stationary phase material described herein, forexample, a fully or superficially porous silica stationary phase bondedwith phenyl moieties, an organosilica stationary phase bonded withphenyl moieties, or a polymeric polystyrene divinyl benzene surfacechemistry. The mobile phase is about 0.005% (v/v) to about 0.20% (v/v)halogenated acid (e.g., DFA) and less than about 100 ppb of anyindividual metal impurity. The instructions instruct the user to obtaina sample of at least one biomolecule (e.g., a protein) in a samplematrix. The user is then instructed to flow the sample with the mobilephase through the column to substantially resolve and retain the atleast one biomolecule (e.g., protein). In addition, the instructionsinstruct the use to detect the at least one biomolecule (e.g., protein)using a detector.

In common practice, an LC mobile phase is prepared by adding a small (10μL to 10 mL) volume of liquid additive, conventionally formic acid ortrifluoroacetic acid, to a desired solvent, such as water oracetonitrile. To that end, it is intended that this technology coverready-to-use mobile phases based on water, acetonitrile, methanol,propanol, butanol, and pentanol (and combinations thereof) modified with0.005 to 0.2% (v/v) halogenated acid (e.g., DFA) (that have less thanabout 100 ppb levels, or less than 50 ppb levels, or less than 20 ppblevels, of individual metal impurities, including but not limited tosodium, potassium, calcium and/or iron), that is purposed for LC-MSanalyses of proteins, wherein the use of such a reagent facilitates theproduction of protein mass spectra having less than 5%, less than 2% orless than 1%, relative intensity corresponding to metal and/or saltadducts, including but not limited to sodium, potassium, calcium and/oriron.

EXAMPLES Example 1: Reversed Phase Chromatography of mAb Subunits

This example compares the effects of three acids, TFA, DFA and formicacid, across four different columns in RPLC systems. Each columnseparated the same reference material. Runs were performed with 0.1%TFA, 0.1% DFA, 0.02% DFA, 0.01% DFA and 0.1% formic acid based mobilephase modifiers. Summarized below is the basic procedure that was usedacross all runs. The results are summarized in FIGS. 1A-1T, and 2A-E.

Reduced, IdeS-digested NIST Reference Material 8671, a humanized IgG1κexpressed from a murine cell line, was obtained in the form of theWaters mAb Subunit Standard (Waters, Milford, Mass.). The contents ofone vial were reconstituted in 0.1% (v/v) formic acid in water. Analysesof this sample were performed using a LC System sold by WatersCorporation (Milford, Mass.) under the tradename ACQUITY® UPLC® H-ClassBio and a separation method outlined below. FIGS. 1A-1T, and 2A-Epresent chromatographic data obtained with several different mobilephase systems used in combination with various RPLC columns. The LCconditions are shown in Table 1 and the Gradient Conditions are shown inTable 2.

TABLE 1 LC Conditions Columns: Waters BioResolve ™ RP mAb Polyphenyl,450 Å 2.7 μm, 2.1 × 50 mm AMT Halo ® Protein C4, 400 Å 3.4 μm, 2.1 × 50mm Agilent AdvanceBio ® RP-mAb Diphenyl, 450 Å 3.5 μm, 2.1 × 50 mmWaters ACQUITY ® UPLC ®, Protein BEH C4, 300 Å 1.7 μm, 2.1 × 50 mmMobile Phase A: 0.01 to 0.1% (v/v) acid in water Mobile Phase B: 0.01 to0.1% (v/v) acid in acetonitrile Column Temperature: 80° C. InjectionVolume: 4 μL Sample Concentration: 0.25 μg/μL Sample Diluent: 0.01 to0.1% (v/v) formic acid in water UV Detection: 280 nm (10 Hz)

TABLE 2 Gradient Table Time Flow Rate (min) (mL/min) % A % B CurveInitial 0.2000 85.0 15.0 Initial 20.00 0.2000 45.0 55.0 6 20.30 0.200020.0 80.0 6 21.30 0.2000 20.0 80.0 6 21.60 0.2000 85.0 15.0 6 25.000.2000 85.0 15.0 6

Example 2: Mass Spectrometry with a Single Quadrupole Mass Detector

This example was done to compare the effect of using DFA and TFA on MSsensitivity based on TIC peak height and TIC peak signal-to-noise ratiowhich are used to define the sensitivity of an LC-MS analysis. Variousmobile phase systems were used to detect the same reference material.Runs were performed with 0.1% TFA, 0.1% DFA, 0.02% DFA, 0.01% DFA and0.1% formic acid mobile phases. Summarized below is the basic procedurethat was used across all runs. The results are summarized in FIGS. 3Aand 3B.

Reduced IdeS-digested NIST Reference Material 8671 was obtained in theform of the Waters mAb Subunit Standard (Waters, Milford, Mass.). Thecontents of one vial was reconstituted in water. Analyses of this samplewere performed using an LC System sold by Waters Corporation (Milford,Mass.) under the tradename Waters ACQUITY® UPLC® H-Class Bio system withUV and MS detectors sold by Waters Corporation (Milford, Mass.),including a Tunable Ultra-Violet (TUV) Detector and an ACQUITY® QDa®Mass Detector. Method conditions were listed as below. FIGS. 3A and 3Bshow the comparison of calculated TIC peak height and signal-to-noiseratio of eluted light chain peak using different mobile phase modifiers.The LC conditions are shown in Table 3, the Gradient Conditions areshown in Table 4, and the MS conditions are shown in Table 5.

TABLE 3 LC Conditions Column: Waters BioResolve ™ RP mAb Polyphenyl, 450Å 2.7 μm, 2.1 × 50 mm Mobile Phase A: 0.02 to 0.1% (v/v) acid in waterMobile Phase B: 0.02 to 0.1% (v/v) acid in acetonitrile ColumnTemperature: 80° C. Injection Volume: 2 μL Sample Concentration: 0.25μg/μL Sample Diluent: Water UV Detection: 280 nm (20 Hz)

TABLE 4 Gradient Table Time Flow Rate (min) (mL/min) % A % B CurveInitial 0.300 95.0 05.0 Initial 10.00 0.300 45.0 55.0 6 10.50 0.300 20.080.0 6 11.50 0.300 20.0 80.0 6 11.51 0.300 95.0 05.0 6 15.00 0.300 95.005.0 6

TABLE 5 MS Conditions Mode: ESI positive Mass Range: 350-1250 m/zCollection Mode: Centroid Cone Voltage: 15 V Probe Temperature: 600° C.Capillary Voltage: 1.5 kV Sample Rate: 2 pts/s

Example 3: High Resolution Mass Spectrometry

This example was done to compare the quality of mass spectra produced bycommercially available DFA and TFA mobile phases. Runs were performedwith 0.1% TFA and 0.1% DFA mobile phases. Summarized below is the basicprocedure that was used across all runs. The results are summarized inFIG. 4.

Reduced, IdeS-digested NIST Reference Material 8671 was obtained in theform of the Waters mAb Subunit Standard (Waters, Milford, Mass.). Thecontents of one vial was reconstituted in water. Analyses of this samplewere performed using an LC System sold by Waters Corporation (Milford,Mass.) under the tradename ACQUITY® UPLC® H-Class Bio system with UV andMS detectors sold by Waters Corporation (Milford, Mass.), including aTunable Ultra-Violet (TUV) Detector and a Synapt® G2-Si QT of MS systemfor detection. FIGS. 4A and 4B demonstrate the different metal adductlevels in deconvoluted mass spectra using the same concentration ofLC-MS grade TFA and reagent grade DFA. The LC conditions are shown inTable 6, the Gradient Conditions for separation with TFA are shown inTable 7, the Gradient Conditions for separation with DFA are shown inTable 8, and the MS conditions are shown in Table 9.

TABLE 6 LC Conditions Column: Waters BioResolve ™ RP mAb Polyphenyl, 450Å 2.7 μm, 2.1 × 50 mm Mobile Phase A: 0.1% (v/v) acid in water MobilePhase B: 0.1% (v/v) acid in acetonitrile Column Temperature: 80° C.Injection Volume: 4 μL Sample Concentration: 0.25 μg/μL Sample Diluent:Water UV Detection: 280 nm (20 Hz)

TABLE 7 Gradient Table for Separation with TFA Time Flow Rate (min)(mL/min) % A % B Curve Initial 0.300 75.0 25.0 Initial 10.00 0.300 55.045.0 6 10.50 0.300 20.0 80.0 6 11.50 0.300 20.0 80.0 6 11.51 0.300 75.025.0 6 15.00 0.300 75.0 25.0 6

TABLE 8 Gradient Table for Separation with DFA Time Flow Rate (min)(mL/min) % A % B Curve Initial 0.300 85.0 15.0 Initial 10.00 0.300 65.035.0 6 10.50 0.300 20.0 80.0 6 11.50 0.300 20.0 80.0 6 11.51 0.300 85.015.0 6 15.00 0.300 85.0 15.0 6

TABLE 9 MS Conditions Mode: ESI positive Mass Range: 500-4000 m/zCollection Mode: Continuum Cone Voltage: 60 V Source Temperature: 120°C. Desolvation Temperature: 450° C. Desolvation Gas: 600 L/h CapillaryVoltage: 2.75 kV Sample Rate: 5 pts/s

Example 4: Analysis of Commercially Available DFA

This example was done to show that commercially available DFA does infact have high levels of metal impurities by quantifying the amount ofmetal impurities by inductively coupled plasma mass spectrometry.

The metals contained within a sample of 10 mL of difluoroacetic acid(Oakwood, part number 001231, lot D06N) were quantified by inductivelycoupled plasma mass spectrometry (ICP-MS). The results of this analysisare provided in FIG. 5, as reported in units of part per billion orng/g. Individual metal concentrations are reported with an uncertaintyof ±50%.

Example 5: Preparing High-Purity DFA

Low metal content high-purity DFA was prepared from the commerciallyavailable DFA by distillation with a PFA (copolymer oftetrafluoroethylene and perfluoroalkyl vinylether) acid purificationsystem sold by Savillex Corporation (Eden Prairie, Minn.) under the nameDST-1000 Acid Purification System. The distillation apparatus was firstreadied for use by passing through 500 mL of commercially available DFA.One and two passes of distillation were thereafter performed to obtainincreasingly pure forms of DFA. (See FIGS. 6A-6C.)

Example 6: Low Abundance Variants for ADC Characterization

The technology is applicable to new methods for mAb and ADC (anti-bodydrug conjugate) subunit analysis. Subunits have been routinelycharacterized by separations with a sub-2 μm C₄-bonded organosilica 300Å fully porous stationary phase, a separation temperature of 80° C.,0.1% TFA mobile phases, and eluent comprised of 90% acetonitrile and 10%isopropanol, the latter being needed to facilitate the recovery ofhydrophobic proteins. A result typical of this method is provided inFIGS. 7A and 7B.

A new technique was developed that simplified the method of FIGS. 7A and7B, accelerated turn-around, and/or improved sensitivity. By using aphenyl-bonded superficially porous stationary phase, higher resolutionand improved selectivity was gained along with a reduction inbackpressure and ability to use faster chromatographic velocities. Withthis change, it was possible to exclude IPA from the mobile phasewithout significantly affecting peak capacity or protein recovery. Alongwith the adoption of DFA, it was also possible to reduce the separationtemperature. This new method is exemplified in FIGS. 7C and 7D. It is ofnote that, while the sub 2 μm column required ultrahigh pressureinstrumentation, the 2.7 μm based approach could be transferable toother less specialized instrumentation by way of having loweroperational back pressures. Furthermore, using the latter platform, itwas possible to optimize nearly all facets of the chromatographicseparation and to facilitate some more strenuous examples of deep-levelcharacterization. Two examples of low abundance variants arediscoverable within the shoulder peaks adjacent to the unmodified Fc/2subunit (See FIGS. 7A-7D). Mass spectra corresponding to these speciesare displayed in FIGS. 8A-8C.

The DFA method (FIG. 8B) produced higher signal-to-noise spectra, whichcould be used to more confidently confirm+16 Da (pre-peak) and +674 Da(post-peak) mass shifts and the corresponding identification of Fcdomain oxidation and an aglycosylated isoform.

CONCLUSIONS

The increasing complexity of biopharmaceutical modalities requires therebe improvements made in analytical methodologies. Reversed-phase liquidchromatography is a powerful technique for the separation of proteinsand/or peptides at all molecular levels, and it becomes inordinatelymore powerful when coupled to mass spectrometry. However, depending onthe use of conventional acid modifiers, such as trifluoroacetic acid(TFA) and formic acid (FA), protein and/or peptide RPLC often exhibitsexcellent chromatographic resolution at the compromise of MS sensitivityor, vice versa, excellent MS sensitivity at the compromise of separationquality. The technology described herein, demonstrates a new choice forLC-MS analyses based on the use of highly purified difluoroacetic acid(DFA) with or without a high-coverage phenyl-bonded superficially porousstationary phase. The use of phenyl-bonded superficially porousstationary phases builds upon the advantages of superficially porousparticles with a unique phenyl bonding that aids in reducing temperatureand ion pairing dependence. This lends itself well to the use of DFA forprotein and/or peptide separations, wherein it has now been shown thatthis alternative ion pairing modifier can reach an optimization betweenchromatography and mass spectrometry otherwise unreachable by TFA andFA.

Purified DFA proved to be key to finding a step change in protein and/orpeptide LC-MS capabilities. As used with the aforementionedhigh-coverage phenyl stationary phase, a DFA-based method greatlyimproved subunit-level profiling of a very hydrophobic, cysteine-linkedauristatin conjugated ADC (see, e.g., Example 6). Moreover, in additionto providing benefits to chromatographic resolution and MS sensitivity,this robust platform was found to greatly increase protein recoverywithout the need to use alcohol co-solvents for elution, even with areduction in column temperature. This is theorized to be an effectresulting from purified DFA being sufficiently acidic so as to minimizeionic secondary interactions (unlike FA) but not as hydrophobic as TFAto force excessively strong adsorption.

While this disclosure has been particularly shown and described withreference to example embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the scope of the technology encompassedby the appended claims.

What is claimed is:
 1. A method of separating an analyte from a sample,the method comprising: flowing a mobile phase through a chromatographycolumn wherein the mobile phase comprises about 0.005% (v/v) to about0.20% (v/v) difluoroacetic acid additive comprising less than about 100ppb of any individual metal impurity and a solvent selected from water,acetonitrile, methanol, propanol, isopropanol, butanol, pentanol, orcombinations thereof, wherein the chromatography column furthercomprises a stationary phase having a phenyl-based surface chemistry;injecting a sample comprising the analyte into the mobile phase; andseparating the analyte from the sample.
 2. The method of claim 1,wherein the chromatography column is a liquid chromatography column. 3.The method of claim 1, wherein the chromatography column is a reversedphase chromatography column.
 4. The method of claim 1, wherein thestationary phase is a superficially porous silica stationary phasebonded with phenyl moieties.
 5. The method of claim 1, wherein thestationary phase is an organosilica stationary phase bonded with phenylmoieties.
 6. The method of claim 1, wherein the mobile phase comprisesless than about 50 ppb of any individual metal impurity.
 7. The methodof claim 1, wherein the mobile phase comprises less than about 20 ppb ofany individual metal impurity.
 8. The method of claim 1, wherein themobile phase comprises about 0.01% (v/v) to about 0.05% (v/v)difluoroacetic acid.
 9. The method of claim 1, wherein the mobile phasecomprises about 0.02% (v/v) to about 0.05% (v/v) difluoroacetic acid.10. The method of claim 1, wherein the mobile phase comprises about0.01% (v/v) to about 0.02% (v/v) difluoroacetic acid.
 11. The method ofclaim 1, wherein the analyte is a protein, a peptide or a combinationthereof.
 12. The method of claim 1, further comprising determining themolecular weight of the analyte.
 13. The method of claim 1, furthercomprising detecting the analyte with a mass spectrometer.
 14. Themethod of claim 13, further comprising generating analyte ions.
 15. Themethod of claim 14, wherein the analyte ions are generated byelectrospray ionization.
 16. The method of claim 13, further comprisingacquiring a mass spectrum of the analyte ions.